Complex molecules, such as peptides, peptide mixtures, polypeptide mixtures, proteins, protein mixtures, biologics and biosimilars (or called follow-on-biologics) especially monoclonal antibodies, are extremely difficult to characterize, group and identify in contrast to small chemical molecules. For example, small molecule drugs are typically composed of only 20 to 100 atoms; small biologics such as hormones are typically composed of 200 to 3000 atoms; while large biologics such as monoclonal antibodies are typically composed of 5,000 to 50,000 atoms.
The terms “Biosimilar” or “Follow-on-Biologic” refer to the products that are claimed to have similar structures and properties to the existing biologic products. Biosimilars are prepared and developed for market approval based on the information of existing biologic products. Due to the high complexity of some biologics, the products produced by different manufacturing processes can only be similar. It is nearly impossible to obtain the products that are exactly the same. Therefore, a powerful analytical method is required to compare, group and identify these complex products.
As generally known, the manufacturing processes for biologics are different from those for small molecule drugs. Small molecule drugs are mainly synthesized via chemical reactions. They can be well-characterized, and can be easily purified and analyzed with routine laboratory tests. The equivalence for small drug products produced by different manufacturing processes can be assessed by simple analytical methods. However, biologics, by comparison, are typically produced within specially engineered cells. Biologics, especially larger biologics, tend to be produced as diverse mixtures of molecules that differ very slightly from one another, which make them difficult to characterize. It follows that the properties of the biologics often depend directly on the nature of the manufacturing process. For example, proteins with unique structural conformation (referred to as “folding”) could express different functions in the body; in addition, biologics that even have the same sequence chemically may have different biological effects due to differences in the structural folding.
One embodiment of the present invention relates to the analytic method for characterizing, comparing and classifying peptides, peptide mixtures, polypeptide mixtures and biomolecules that comprise a polypeptide component by mass spectrometry.
Copolymer-1 is a complex mixture of polypeptides prepared from the polymerization of the amino acids glutamic acid, lysine, alanine and tyrosine. Copolymer-1 also is known as glatiramer acetate and has the following structural formula:(Glu,Ala,Lys,Tyr)χ.χCH3COOH(C5H9NO4.C3H7NO2.C6H14N2O2.C9H11NO3)χ.χC2H4O2 (Physician Desk Reference, (2000))
Glatiramer acetate (GA) is the active ingredient of COPAXONE® (Teva Pharmaceutical Industries Ltd., Israel), which comprises the acetate salts of a synthetic polypeptide mixture containing four naturally occurring amino acids: L-glutamic acid, L-alanine, L-tyrosine, and L-lysine, with a reported average molar fraction of 0.141, 0.427, 0.095, and 0.338, respectively. The average molecular weight of COPAXONE® is between 4,700 and 11,000 daltons. Glatiramer acetate is an approved drug for the treatment of multiple sclerosis (MS). Processes for the preparation of glatiramer acetate are described in U.S. Pat. Nos. 3,849,550 and 5,800,808 and PCT International Publication No. WO 00/05250.
European Patent Application Publication No. 1 983 344 A1 discloses a method for digesting a single polypeptide standard by Trypsin and detecting its fragmentation by MADLDI-TOF. PCT International Publication No. WO 2008/135756 discloses digesting a single peptide standard by Trypsin, which provided the expected tryptic peptide fragments to be analyzed by tandem MS.